ifitm3 human  (Eurofins)

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3. (A) Endogenous MYCT1 is located at cell–cell junctions (arrow) and in puncta (arrowhead). Staining of human primary ECs for MYCT1 (black), VE-cadherin (magenta), and DNA (blue). Scale bar, 10 µm. (B) MYCT1 is a membrane protein. Western blot analysis of various EC fractions for MYCT1, GAPDH, PECAM1, H3K27ac, and vimentin proteins. Cy, cytoplasm; Mb, membrane; Nu, nucleus; Ck, cytoskeleton. (C) MYCT1 is glycosylated. Western blot analysis of MYCT1 protein electrophoretic mobility in control and PNGase-F–treated lysates. (D) Schematic model of MYCT1 structure and domains with phosphorylation sites, identified by mass spectrometry. MYCT1 phosphorylation sites are highly conserved as indicated by the color scale. Asterisks indicate sites also described at https://www.phosphosite.org/ . (E) Top five proteins interacting with MYCT1 as identified by mass spectrometry, among which IFITM2 and IFITM3. ECs were transduced with recombinant adenoviruses to transiently overexpress MYCT1 or GFP, as a control. Cell lysates were collected 48 h after transduction, immunoprecipitated using MYCT1 antibody or a control IgG, and analyzed by mass spectrometry. Proteins interacting with both endogenous and overexpressed MYCT1 were selected and ranked by normalized spectral abundance factor (NSAF) from two independent mass spectrometry (MS) experiments are shown (31 proteins); the top five proteins are highlighted in magenta. (F) GO terms of the cellular component and biological process overrepresented in the MYCT1 interactome. Fisher’s exact test with adjustment for false discovery rate (FDR). (G) Validation of IFITM2/3 and MYCT1 interaction by co-IP. EC lysates from confluent ECs were immunoprecipitated (IP) with MYCT1 or control IgG and blotted for IFITM2/3. H, IgG heavy chain; L, IgG light chain. (H) IFITM2/3 are constitutively expressed in ECs in vitro and in vivo . Staining of human primary ECs (upper panels), human brain and WAT sections (lower panels) for MYCT1 (gray), IFITM2/3 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm (brain) and 50 µm (adipose tissue). (I) MYCT1 and IFITM2/3 interact in brain ECs. Proximity ligation assay (PLA) in human brain sections. Detection of PLA dots (gray) in ECs and staining for VE-cadherin (magenta) and DNA (blue). Arrowheads, colocalization of MYCT1::IFITM2/3 PLA dots and VE-cadherin staining. Scale bar, 20 µm. See also . Source data are available for this figure: .

Article Snippet: IFITM3 (human) forward , Eurofins , 5′-ACC ATG AAT CAC ACT GTC CAA ACC TT-3′.

Techniques: Staining, Membrane, Western Blot, Control, Phospho-proteomics, Mass Spectrometry, Transduction, Recombinant, Immunoprecipitation, Biomarker Discovery, Co-Immunoprecipitation Assay, In Vitro, In Vivo, Proximity Ligation Assay

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 restricts endothelial endocytosis and IFITM2/3-dependent mTORC1 activation, related to Figs. 6 and 7. (A) IFITM2/3 antibody and siRNA validation for identification of endogenous human IFITM2/3 proteins. IFITM2/3 knockdown reduces MYCT1 protein levels. Staining of ECs for MYCT1 (gray), IFITM2/3 (green), and DNA (blue). Scale bar, 20 µm. (B) Quantification of MYCT1 protein levels in control and IFITM2/3 KD cells. n = 4 independent experiments; mean ± SD; Welch’s t test, P = 0.0014 (*). (C and D) MYCT1 knockdown does not affect IFITM2 (C) nor IFITM3 (D) mRNA levels in ECs. n = 3 independent experiments; mean ± SD; Welch’s t test, P > 0.05. (E) MYCT1 knockdown does not impact RAB7 + late endosomes nor LAMP1 + endolysosomes. Staining of ECs for RAB7 (gray), LAMP1 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm. (F and G) Quantification of RAB7 + (F) and LAMP1 + (G) areas per cell in control and MYCT1 KD cells. n = 3 independent experiments; 20–50 cells were analyzed per condition for each experiment; mean ± SD; Welch’s t test, P > 0.05. (H) MYCT1 knockdown increased FITC-dextran uptake. 2 days after siRNA transfection, cells were starved for 1 h in PBS, followed by a 30-min induction with amino acid solution together with 10-kDa FITC dextran. Detection of 10-kDa FITC-dextran (gray) and staining of ECs for VE-cadherin (magenta) and DAPI (blue). Arrow, dextran + puncta. Scale bar, 10 μm. (I) Quantification of the number of dextran + puncta per cell in control and MYCT1 KD cells. n = 3 independent experiments; 30–50 cells were analyzed per condition for each experiment; mean ± SD; Welch’s t test, P = 0.0016 (*). (J) Example of gating strategy (7-AAD neg CD45 neg CD31 + ) of ECs from gonadal fat pad by flow cytometry. (K) WAT ECs take up higher amounts of labeled plasma proteins compared with colon ECs. Quantification of labeled plasma protein uptake in ECs from s.c. and visceral WAT and colon normalized to plasma Atto-647 signal. n = 10 mice per organ; Friedman test with Dunn’s multiple comparisons test, P > 0.05 for scFAT versus visFAT, P = 0.0052 for scFAT versus colon, and P = 0.001 (*) for visFAT versus colon. (L) Endocytosis inhibition with dynasore rescues mTORC1 hyperactivation caused by knockdown of MYCT1 . Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 50 μm. (M) Quantification of mTORC1 activation by amino acid supplementation in control and MYCT1 KD cells in the absence or presence of dynasore. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 3 independent experiments; 1,500–6,000 cells were analyzed per condition for each experiment; mean ± SD; two-way ANOVA with Tukey’s multiple comparisons test, P = 0.004 (*) for MYCT1 knockdown effect in control conditions and P < 0.001 (*) for its rescue by dynasore treatment. (N) RAB5 knockdown rescues mTORC1 hyperactivation in MYCT1 KD cells. Staining of ECs for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 50 μm. (O) Quantification of mTORC1 activation in control, MYCT1 KD , and MYCT1-RAB5 KD cells. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 3 independent experiments; 7,000-15,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0021 (*) for MYCT1 knockdown effect and P = 0.0292 (*) for its rescue by RAB5 double knockdown. (P) IFITM2/3 knockdown rescues mTORC1 hyperactivation in MYCT1 -deficient human adipose ECs. Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 100 μm. (Q) Quantification of mTORC1 activation in control, MYCT1 KD , IFITM2/3 KD , and MYCT1 – IFITM2/3 KD cells. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 2 independent experiments; 1,500–3,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0168 (*) for MYCT1 knockdown effect and P = 0.0123 (*) for rescue effect by IFITM2/3 double knockdown.

Article Snippet: IFITM3 (human) forward , Eurofins , 5′-ACC ATG AAT CAC ACT GTC CAA ACC TT-3′.

Techniques: Activation Assay, Biomarker Discovery, Knockdown, Staining, Control, Transfection, Flow Cytometry, Labeling, Clinical Proteomics, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3. (A) Endogenous MYCT1 is located at cell–cell junctions (arrow) and in puncta (arrowhead). Staining of human primary ECs for MYCT1 (black), VE-cadherin (magenta), and DNA (blue). Scale bar, 10 µm. (B) MYCT1 is a membrane protein. Western blot analysis of various EC fractions for MYCT1, GAPDH, PECAM1, H3K27ac, and vimentin proteins. Cy, cytoplasm; Mb, membrane; Nu, nucleus; Ck, cytoskeleton. (C) MYCT1 is glycosylated. Western blot analysis of MYCT1 protein electrophoretic mobility in control and PNGase-F–treated lysates. (D) Schematic model of MYCT1 structure and domains with phosphorylation sites, identified by mass spectrometry. MYCT1 phosphorylation sites are highly conserved as indicated by the color scale. Asterisks indicate sites also described at https://www.phosphosite.org/ . (E) Top five proteins interacting with MYCT1 as identified by mass spectrometry, among which IFITM2 and IFITM3. ECs were transduced with recombinant adenoviruses to transiently overexpress MYCT1 or GFP, as a control. Cell lysates were collected 48 h after transduction, immunoprecipitated using MYCT1 antibody or a control IgG, and analyzed by mass spectrometry. Proteins interacting with both endogenous and overexpressed MYCT1 were selected and ranked by normalized spectral abundance factor (NSAF) from two independent mass spectrometry (MS) experiments are shown (31 proteins); the top five proteins are highlighted in magenta. (F) GO terms of the cellular component and biological process overrepresented in the MYCT1 interactome. Fisher’s exact test with adjustment for false discovery rate (FDR). (G) Validation of IFITM2/3 and MYCT1 interaction by co-IP. EC lysates from confluent ECs were immunoprecipitated (IP) with MYCT1 or control IgG and blotted for IFITM2/3. H, IgG heavy chain; L, IgG light chain. (H) IFITM2/3 are constitutively expressed in ECs in vitro and in vivo . Staining of human primary ECs (upper panels), human brain and WAT sections (lower panels) for MYCT1 (gray), IFITM2/3 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm (brain) and 50 µm (adipose tissue). (I) MYCT1 and IFITM2/3 interact in brain ECs. Proximity ligation assay (PLA) in human brain sections. Detection of PLA dots (gray) in ECs and staining for VE-cadherin (magenta) and DNA (blue). Arrowheads, colocalization of MYCT1::IFITM2/3 PLA dots and VE-cadherin staining. Scale bar, 20 µm. See also . Source data are available for this figure: .

Article Snippet: IFITM3 (human) reverse , Eurofins , 5′-CCA GCA CAG CCA CCT CG-3′.

Techniques: Staining, Membrane, Western Blot, Control, Phospho-proteomics, Mass Spectrometry, Transduction, Recombinant, Immunoprecipitation, Biomarker Discovery, Co-Immunoprecipitation Assay, In Vitro, In Vivo, Proximity Ligation Assay

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 restricts endothelial endocytosis and IFITM2/3-dependent mTORC1 activation, related to Figs. 6 and 7. (A) IFITM2/3 antibody and siRNA validation for identification of endogenous human IFITM2/3 proteins. IFITM2/3 knockdown reduces MYCT1 protein levels. Staining of ECs for MYCT1 (gray), IFITM2/3 (green), and DNA (blue). Scale bar, 20 µm. (B) Quantification of MYCT1 protein levels in control and IFITM2/3 KD cells. n = 4 independent experiments; mean ± SD; Welch’s t test, P = 0.0014 (*). (C and D) MYCT1 knockdown does not affect IFITM2 (C) nor IFITM3 (D) mRNA levels in ECs. n = 3 independent experiments; mean ± SD; Welch’s t test, P > 0.05. (E) MYCT1 knockdown does not impact RAB7 + late endosomes nor LAMP1 + endolysosomes. Staining of ECs for RAB7 (gray), LAMP1 (green), VE-cadherin (magenta), and DNA (blue). Scale bar, 20 µm. (F and G) Quantification of RAB7 + (F) and LAMP1 + (G) areas per cell in control and MYCT1 KD cells. n = 3 independent experiments; 20–50 cells were analyzed per condition for each experiment; mean ± SD; Welch’s t test, P > 0.05. (H) MYCT1 knockdown increased FITC-dextran uptake. 2 days after siRNA transfection, cells were starved for 1 h in PBS, followed by a 30-min induction with amino acid solution together with 10-kDa FITC dextran. Detection of 10-kDa FITC-dextran (gray) and staining of ECs for VE-cadherin (magenta) and DAPI (blue). Arrow, dextran + puncta. Scale bar, 10 μm. (I) Quantification of the number of dextran + puncta per cell in control and MYCT1 KD cells. n = 3 independent experiments; 30–50 cells were analyzed per condition for each experiment; mean ± SD; Welch’s t test, P = 0.0016 (*). (J) Example of gating strategy (7-AAD neg CD45 neg CD31 + ) of ECs from gonadal fat pad by flow cytometry. (K) WAT ECs take up higher amounts of labeled plasma proteins compared with colon ECs. Quantification of labeled plasma protein uptake in ECs from s.c. and visceral WAT and colon normalized to plasma Atto-647 signal. n = 10 mice per organ; Friedman test with Dunn’s multiple comparisons test, P > 0.05 for scFAT versus visFAT, P = 0.0052 for scFAT versus colon, and P = 0.001 (*) for visFAT versus colon. (L) Endocytosis inhibition with dynasore rescues mTORC1 hyperactivation caused by knockdown of MYCT1 . Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 50 μm. (M) Quantification of mTORC1 activation by amino acid supplementation in control and MYCT1 KD cells in the absence or presence of dynasore. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 3 independent experiments; 1,500–6,000 cells were analyzed per condition for each experiment; mean ± SD; two-way ANOVA with Tukey’s multiple comparisons test, P = 0.004 (*) for MYCT1 knockdown effect in control conditions and P < 0.001 (*) for its rescue by dynasore treatment. (N) RAB5 knockdown rescues mTORC1 hyperactivation in MYCT1 KD cells. Staining of ECs for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 50 μm. (O) Quantification of mTORC1 activation in control, MYCT1 KD , and MYCT1-RAB5 KD cells. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 3 independent experiments; 7,000-15,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0021 (*) for MYCT1 knockdown effect and P = 0.0292 (*) for its rescue by RAB5 double knockdown. (P) IFITM2/3 knockdown rescues mTORC1 hyperactivation in MYCT1 -deficient human adipose ECs. Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 100 μm. (Q) Quantification of mTORC1 activation in control, MYCT1 KD , IFITM2/3 KD , and MYCT1 – IFITM2/3 KD cells. The percentage of p-S6 + cells was quantified in the indicated conditions. n = 2 independent experiments; 1,500–3,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0168 (*) for MYCT1 knockdown effect and P = 0.0123 (*) for rescue effect by IFITM2/3 double knockdown.

Article Snippet: IFITM3 (human) reverse , Eurofins , 5′-CCA GCA CAG CCA CCT CG-3′.

Techniques: Activation Assay, Biomarker Discovery, Knockdown, Staining, Control, Transfection, Flow Cytometry, Labeling, Clinical Proteomics, Inhibition

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Analyzing the knockdown of IFITM3 efficiency in LV-shIFITM3 KG-1a cells. (A) The efficiency of lentivirus infection of the KG-1a cells was observed under a conventional fluorescent microscope. (B) The IFITM3 mRNA expression in the KG-1a cells was detected by qRT-PCR. (C) The expression of the IFITM3 protein in the KG-1a cells was detected by Western blot. (D) The results from the semi-quantitative analysis of protein expression levels. ****P<0.00001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Knockdown, Infection, Microscopy, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Down-regulation of IFITM3 changed the biological properties of the KG-1a cells. (A) The effect of down-regulated IFITM3 on the proliferation ability of KG-1a cells as detected by CCK8 assay. (B) The colony formations of various KG-1a cells detected by soft agar cloning assay (50×); (C) Comparisons of the colony formations; (D) The FCM analysis results of the KG-1a cell cycle changes; (E) Comparisons of the cell cycles; (F) The effects of Ara-C (0.4μM) on the apoptosis of various KG-1a cells analyzed by FCM (24h). (G) The FCM results; (H) The mRNA expression level of caspase 3 in various KG-1a cells as detected by qRT-PCR. *P < 0.05, ***P < 0.001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: CCK-8 Assay, Cloning, Expressing, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: The IFITM3 expression and the proliferation of the KG-1a cells. (A) The IFITM3 expression as detected by Western blot after the KG-1a cells were treated with PBS, PBNPs and IONPs, respectively, for 72h; (B) The semi-quantitative analysis of the Western blot results; (C) The IFITM3 expression in the KG-1a cells as detected by Western blot after the cells were treated with PBS, PBNPs and IONPs, respectively, for 72h; (D) The proliferation of the KG-1a cells as detected by the CCK8 assay in the KG-1a cells that were treated with different drugs for 96h; (E) The qRT-PCR-detected expression level of c-myc in the KG-1a cells treated with different drugs for 24h. *P < 0.05, **P < 0.01, ***P < 0.001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Expressing, Western Blot, CCK-8 Assay, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Impact of IFITM3 knockdown on disease progression in AML model mice. (A) Leukemia cells in peripheral blood and bone marrow; (B) With Reichsen-Giemsa staining; (C) Leukemia cells of bone marrow (left: under a light microscope; right: under a fluorescence microscope) in the AML-bearing mice injected with the Scrambled or the Lv-shIFITM3-KG1a cells; (D) The expression levels of CD33, CD123 and CD11b detected by FCM. (E) Statistical results of the CD33, CD123 and CD11b expression levels. The experiment was repeated twice. *P < 0.05, ***P < 0.001.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Knockdown, Biomarker Discovery, Staining, Light Microscopy, Fluorescence, Microscopy, Injection, Expressing

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Identification of host factors essential for PEDV infection via genome-wide CRISPR/Cas9 screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.

Article Snippet: A polyclonal antibody for human IFITM3 (cat#: 11714-1-AP) was obtained from Proteintech.

Techniques: Infection, Genome Wide, CRISPR, Expressing, Transduction, Amplification, Next-Generation Sequencing, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Endogenous IFITM3 expression enhances PEDV infection. ( A ) Huh7 and Huh7.5 cells were inoculated with the PEDV SD virus at MOIs of 0.01, 0.05, and 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining. Representative fluorescence microscopy images of the intracellular PEDV N protein in Huh7 and Huh7.5 cells. Red indicates the viral N protein, and blue denotes the cell nuclei (DAPI). Scale bar: 200 µm. ( B ) Western blot analysis revealed the protein levels of PEDV N protein and IFITM3 in both the Huh7 and Huh7.5 cell lines after infection. GAPDH served as an internal control. ( C ) The viral genomic RNA in the supernatants was quantified via qRT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the s.d. from three technical replicates ( n = 3). ****, P < 0.0001. ( D ) The number of infectious PEDV particles in the supernatants of Huh7 and Huh7.5 cells infected with different MOIs of PEDV SDs was assessed via the TCID 50 assay. The error bars indicate the s.d. from three technical experiments. ****, P < 0.0001. ( E ) The protein expression of PEDV N protein and IFITM3 in Huh7.5 cells treated with different doses of IFN-β was analyzed by Western blotting. The relative expressions of the PEDV N protein were normalized against GAPDH, as indicated by the numerical values displayed below the blot. ( F ) Huh7.5 cells were treated with various doses of IFN-β for 12 h and then infected with 0.01 MOI of PEDV. Virus titers in the supernatant were analyzed via the TCID 50 assay. *, P < 0.05; ***, P < 0.001.

Article Snippet: A polyclonal antibody for human IFITM3 (cat#: 11714-1-AP) was obtained from Proteintech.

Techniques: Expressing, Infection, Virus, Immunofluorescence, Staining, Fluorescence, Microscopy, Western Blot, Control, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Both IFITM2 and IFITM3 enhance PEDV infection. ( A ) IFITM3 was reintroduced into IFITM3-KO Huh7 cells via transduction with a lentivirus expressing IFITM3 or a control empty vector, after which the cells were infected with the PEDV SD strain. At 24 h post-infection, the levels of the IFITM3 protein and PEDV N protein were analyzed via Western blotting. ( B ) The viral titers in the supernatants of the various cells in ( A ) were assessed via the TCID 50 assay. ****, P < 0.0001. ( C ) Huh7.5 cells were transduced with lentiviral vectors expressing either IFITM2 or IFITM3 or a control empty vector, followed by PEDV infection. The expression of the PEDV N protein and IFITM protein was assessed by Western blot analysis. ( D ) The expression of the PEDV N protein from ( C ) was also visualized via immunofluorescence staining. Representative images of the intracellular PEDV N protein. Red indicates the viral N protein, and blue denotes the cell nuclei (DAPI). Scale bar: 200 µm. ( E ) Huh7.5 cells were transduced with lentiviral vectors expressing either IFITM2 or IFITM3 or a control empty vector, followed by PEDV infection. The viral titers in the supernatant were measured via the TCID 50 assay. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: A polyclonal antibody for human IFITM3 (cat#: 11714-1-AP) was obtained from Proteintech.

Techniques: Infection, Transduction, Expressing, Control, Plasmid Preparation, Western Blot, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: IFITM3 enhances PEDV entry into host cells. ( A ) IFITM3-WT and IFITM3-KO Huh7 cells were infected with equal amounts of the PEDV spike pseudotyped virus rVSV-ΔG-EGFP-PEDV-S. Samples were collected at 12 h post-infection (hpi) and analyzed via fluorescence microscopy and flow cytometry. Scale bars: 200 µm. ( B ) The relative transduction rates obtained from the flow cytometry analysis in ( A ) were quantified. The data are expressed as the means ± s.d. of three independent experiments. ****, P < 0.0001. ( C ) RT-qPCR data showing the relative expression of IFITM3 mRNA in Huh7 cells transduced with lentiviral vectors carrying either IFITM3 shRNAs or scramble shRNAs (control). IFITM3 expression in control cells was normalized to 1. The values are presented as the means ± s.d., n = 3. ( D ). Representative Western blots showing the levels of the PEDV N protein (top), IFITM3 protein (middle), and GAPDH (bottom; loading control) in Huh7 cells transduced with lentiviral vectors containing IFITM3 shRNAs or scramble shRNA. ( E ) IFITM3-knockdown cells were infected with the same amount of rVSV-ΔG-EGFP-PEDV-S. At 12 hpi, the efficiency of virus transduction was assessed via fluorescence microscopy and flow cytometry. ( F ) The relative transduction rates of rVSV-ΔG-EGFP-PEDV-S in IFITM3-knockdown Huh7 cells were analyzed via flow cytometry. The values are presented as the means ± s.d. ( n = 3). ****, P < 0.0001. ( G ) A comparison of the transduction rates between Huh7 and Huh7.5 cells infected with equal amounts of rVSV-ΔG-EGFP-PEDV-S is shown. ( H ) Flow cytometry analysis of IFITM3-KO Huh7 cells re-expressing IFITM3 and infected with the same amount of rVSV-ΔG-EGFP-PEDV-S. ( I ) Flow cytometry analysis of Huh7.5 cells overexpressing either IFITM3 or IFITM2 and infected with equal amounts of rVSV-ΔG-EGFP-PEDV-S. All data are presented as the means ± s.d. from three independent experiments.

Article Snippet: A polyclonal antibody for human IFITM3 (cat#: 11714-1-AP) was obtained from Proteintech.

Techniques: Infection, Virus, Fluorescence, Microscopy, Flow Cytometry, Transduction, Quantitative RT-PCR, Expressing, Control, Western Blot, shRNA, Knockdown, Comparison

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Impact of IFITM3 on PEDV attachment, internalization, and viral RNA replication. ( A ) Huh7.5-IFITM3 and Huh7.5-vector cells were infected with PEDV at an MOI of 10 for 1 h on ice. The cells were subsequently washed three times with cold DPBS, and viral attachment to the cell surface was quantified via RT-qPCR. ( B ) Following viral attachment for 1 h on ice, the cells were incubated at 37°C for an additional 1 h and viral internalization was assessed by RT-qPCR. ( C ) Huh7.5-IFITM3 and Huh7.5-vector cells, as well as ( D ) IFITM3-WT and IFITM3-KO cells, were infected with PEDV at an MOI of 10 at 37°C for 1 h. After the inoculum was removed, the cells were either immediately harvested or cultured at 37°C for the specified time points. The amounts of cell-associated viral RNA were quantified via a qRT-PCR assay and are presented as the copy number per 100 ng of total RNA. The data are presented as the means ± s.d. from three independent experiments.

Article Snippet: A polyclonal antibody for human IFITM3 (cat#: 11714-1-AP) was obtained from Proteintech.

Techniques: Plasmid Preparation, Infection, Quantitative RT-PCR, Incubation, Cell Culture

Journal: Viruses

Article Title: Common Chemical Plasticizer Di(2-Ethhylhexyl) Phthalate Exposure Exacerbates Coxsackievirus B3 Infection

doi: 10.3390/v16121821

Figure Lengend Snippet: DEHP exacerbates CVB infection by enhancing interferon-induced transmembrane 2 and 3 in HeLas and iBECs. ( A , B ) Western blots detecting IFITM2/3 in HeLa ( A ) and iBECs ( B ) treated with vehicle or DEHP prior to infecting with EGFP-CVB or mock-infecting. Pon S stain is shown below. Densitometric quantification of IFITM2/3 is shown to the right. ( C ) Fluorescence microscopy detecting enhanced green fluorescent protein (EGFP) expression between cells transfected with scramble siRNA ( siSCRAMBLE) (top), IFITM2 siRNA ( siIFITM2 ) (middle), or IFITM3 siRNA ( siIFITM3 ) (bottom) for 48 h prior to infecting with EGFP-CVB at MOI of 0.001, respectively, for 24 h (scale bars = 100 µm). ( D ) Western blots detecting VP1 and IFITM2/3 in HeLas treated with siIFITM2 for 48 h before infecting with EGFP-CVB at MOI of 0.001, respectively, for 24 h. Pon S stain is shown below. Densitometric quantification of VP1 and IFITM2/3 is shown to the right. ( E ) Western blots detecting VP1 and IFITM3 in HeLas treated with siIFITM3 for 48 h before infecting with EGFP-CVB at MOI of 0.001, respectively, for 24 h. Pon S stain is shown below. Densitometric quantification of VP1 and IFITM2/3 is shown to the right. ( F ) Viral plaque assays enumerating infectious viral titers in tissue culture media from cells in C. (Student’s t-test ( D , E ), ANOVA ( A , B , D – F ); n = 3 per group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars represent standard deviation.)

Article Snippet: Scrambled siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-37007), human IFITM2 siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-96760), and human IFITM3 siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-97053) were reconstituted in nuclease-free water following the manufacturer-provided datasheet. siRNAs were transfected into HeLa cells using an Effectene Transfection Reagent (Qiagen, Valencia, CA, USA, 301425) according to the manufacturer’s guidelines for reagent volumes.

Techniques: Infection, Western Blot, Staining, Fluorescence, Microscopy, Expressing, Transfection, Standard Deviation

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 1. Interferon-induced transmem- brane protein 3 (IFITM3) expression in the normal kidney and clear cell renal cell carcinoma (ccRCC). (A) In normal renal parenchyma, IFITM3 was immuno- reactive in glomerular parietal epithelial cells, capillaries (left), and damaged or atrophic tubules (right). IFITM3 ex- pression was inconspicuous in tubular epithelial cells (middle). (B) In ccRCC, IFITM3 was immunoreactive as a mem- branous pattern in low-grade tumors (left), whereas cytoplasmic patterns are observed in high-grade tumors (right) (magnification, ×400).

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Expressing

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 3. (A) The tumorigenicity of 786-O P/empty, 786-O suR/empty, 786-O P/IFITM3-overexpression, and 786-O suR/IFITM3-shRNA was evaluated in a xenograft model. Sunitinib was administered 31 days after transplantation. (B) Representative images 786-O P/IFITM3-overexpression (left upper), 786-O P/empty, 786-O suR/IFITM3-shRNA, and 786-O suR/empty were presented clockwise. Representative images of the high-power field were presented as inset. IFITM3, interferon-induced transmembrane protein 3 (magnification, ×40; ×400).

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Over Expression, shRNA, Transplantation Assay

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 5. The interferon-induced trans- membrane protein 3 (IFITM3)-induced acquired tyrosine kinase inhibitor (TKI) resistance mechanism represented in a schematic diagram. In patients resistant to sunitinib, overexpression of IFITM3 is observed. The binding of IFITM3 to TRAF6 leads to the activation of TRAF6 associated with IRAK1, which induces oncogenic signaling pathways, includ- ing MAPK/AP-1 pathways. As a result, clear cell renal cell carcinoma (ccRCC) can accelerate tumor growth, survival, and proliferation despite TKI treatment.

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Membrane, Over Expression, Binding Assay, Activation Assay, Protein-Protein interactions